Bottom Line:
We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2.Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I).We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

ABSTRACTDuring anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

Figure 1: The control G1 cells released at 34°C. (A) Photomicrographs taken 105 min after release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and shown in blue. (B) FACS® profile shows that for these cells the time between DNA replication and cytokinesis is ∼75 min. (C) Sister chromatids separate 30 min after budding. 22% of the cells have long spindles in anaphase. Bar, 5 μm.

Mentions:
We examined the role of Stu2 during anaphase by using the temperature-sensitive allele stu2-10. We obtained a population of stu2-10 G1 cells at room temperature using centrifugal elutriation and released them into fresh medium at 37°C. The cells arrested with short spindles, 2C DNA, and nonseparated sister chromatids, showing that mutants in stu2-10 generate a metaphase arrest (data not shown). To minimize complications arising from destabilization of spindles at this high temperature, we determined the minimal restrictive temperature for stu2-10 by shifting a cycling population of stu2-10 cells to various temperatures. This analysis showed that the minimal nonpermissive temperature for stu2-10 cells was 34°C (data not shown). To confirm this result, we grew stu2-10 cells at room temperature, obtained a population of G1 cells by centrifugal elutriation, and released them at 34°C. Although control cells produced long spindles by 105 min after the release (Fig. 1A and Fig. C), stu2-10 cells never elongated their spindles (Fig. 2A and Fig. C). To quantify spindle elongation, we counted the percentage of spindles that had completed anaphase B (long spindles) throughout the release. Spindles of a length greater than the diameter of the mother cell were counted as long. As shown in Fig. 2 C, at 90 min after the release, <1% of total number of stu2-10 cells had long spindles, whereas in the population of the control cells the proportion of the cells with long spindles is 22% (Fig. 1 C). FACS® analysis showed that stu2-10 cells delay cytokinesis (Fig. 2 B) by 45 min compared with the control cells (Fig. 1 B), suggesting that stu2-10 delays progression from metaphase to anaphase. To further analyze the cell cycle state in stu2-10 mutants, we analyzed sister chromatid separation throughout the release by introducing a CENV–GFP construct (Michaelis et al. 1997) into all the yeast strains described in this study. Although control cells start to separate sister chromatids 45 min after budding (Fig. 1 C), stu2-10 mutants start sister chromatid separation at ∼90 min after budding (Fig. 2 C), confirming the existence of a significant metaphase delay in stu2-10.

Figure 1: The control G1 cells released at 34°C. (A) Photomicrographs taken 105 min after release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and shown in blue. (B) FACS® profile shows that for these cells the time between DNA replication and cytokinesis is ∼75 min. (C) Sister chromatids separate 30 min after budding. 22% of the cells have long spindles in anaphase. Bar, 5 μm.

Mentions:
We examined the role of Stu2 during anaphase by using the temperature-sensitive allele stu2-10. We obtained a population of stu2-10 G1 cells at room temperature using centrifugal elutriation and released them into fresh medium at 37°C. The cells arrested with short spindles, 2C DNA, and nonseparated sister chromatids, showing that mutants in stu2-10 generate a metaphase arrest (data not shown). To minimize complications arising from destabilization of spindles at this high temperature, we determined the minimal restrictive temperature for stu2-10 by shifting a cycling population of stu2-10 cells to various temperatures. This analysis showed that the minimal nonpermissive temperature for stu2-10 cells was 34°C (data not shown). To confirm this result, we grew stu2-10 cells at room temperature, obtained a population of G1 cells by centrifugal elutriation, and released them at 34°C. Although control cells produced long spindles by 105 min after the release (Fig. 1A and Fig. C), stu2-10 cells never elongated their spindles (Fig. 2A and Fig. C). To quantify spindle elongation, we counted the percentage of spindles that had completed anaphase B (long spindles) throughout the release. Spindles of a length greater than the diameter of the mother cell were counted as long. As shown in Fig. 2 C, at 90 min after the release, <1% of total number of stu2-10 cells had long spindles, whereas in the population of the control cells the proportion of the cells with long spindles is 22% (Fig. 1 C). FACS® analysis showed that stu2-10 cells delay cytokinesis (Fig. 2 B) by 45 min compared with the control cells (Fig. 1 B), suggesting that stu2-10 delays progression from metaphase to anaphase. To further analyze the cell cycle state in stu2-10 mutants, we analyzed sister chromatid separation throughout the release by introducing a CENV–GFP construct (Michaelis et al. 1997) into all the yeast strains described in this study. Although control cells start to separate sister chromatids 45 min after budding (Fig. 1 C), stu2-10 mutants start sister chromatid separation at ∼90 min after budding (Fig. 2 C), confirming the existence of a significant metaphase delay in stu2-10.

Bottom Line:
We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2.Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I).We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

ABSTRACTDuring anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.